This report describes an integrated and modular microsystem providing rapid analyses of trace-level tryptic digests for proteomics applications. This microsystem includes an autosampler, a microfabricated device comprising a large channel (2.4 µL total volume), an array of separation channels together with a low dead volume enabling the interface to nanoelectrospray mass spectrometry. The large channel of this microfluidic device provides a convenient platform to integrate C18 reverse phase packing or other type of affinity media such as immobilized antibodies or IMAC beads thus enabling affinity selection of target peptides prior to electrophoretic separation and mass spectrometry analyses on a quadrupole/time-of-flight instrument. Sequential injection, preconcentration and separation of peptide standards and tryptic digests are achieved with a throughput of up to 12 samples/per hour and a concentration detection limit of approximately 20 nM. Replicate injections of peptide mixtures indicated that reproducibility of migration time was 1.2-1.8 %, whereas RSD ranging from 9.2-11.8 % are observed on peak heights. The application of this device for trace level protein identification is demonstrated for 2-D gel spots obtained from extracts of human prostatic cancer cells (LNCap) using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry. Enrichment of target peptides prior to mass spectral analyses is achieved using c-myc specific antibodies immobilized on protein G sepharose beads and facilitated the identification of antigenic peptides spiked at a level of 20ng/mL in human plasma. Affinity selection is also demonstrated for gel-isolated protein bands where tryptic phosphopeptides are first captured on IMAC beads and subsequently separated and characterized on this microfluidic system.